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Home , Ascoviridae, Mamavirus, Megavirus

Vol. 102: 195–209, 2013 DISEASES OF AQUATIC ORGANISMS Published February 28 doi: 10.3354/dao02548 Dis Aquat Org

A new species of nucleo-cytoplasmic large DNA (NCLDV) associated with mortalities in Manitoba lake sturgeon Acipenser fulvescens

Sharon C. Clouthier1,*, Elissa VanWalleghem1,2, Shelagh Copeland3, Cheryl Klassen2, Gary Hobbs4, Ole Nielsen1, Eric D. Anderson5

1Freshwater Institute, Fisheries & Oceans Canada, 501 University Crescent, Winnipeg, Manitoba R3T 2N6, Canada 2Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada 3Manitoba Agriculture, Food and Rural Initiatives, Veterinary Diagnostic Services, 545 University Crescent, Winnipeg, Manitoba R3T 5S6, Canada 4Manitoba Conservation and Water Stewardship, Ecological Services Division, Grand Rapids, Manitoba R0C 1E0, Canada 5Box 28, Group 30, RR2, Ste. Anne, Manitoba R5H 1R2, Canada

ABSTRACT: A newly discovered virus, Namao virus, associated with morbidity and mortality, was detected among juvenile lake sturgeon Acipenser fulvescens being propagated by a conservation stocking program for this endangered species in Manitoba, Canada. The outbreaks resulted in cumulative mortalities of 62 to 99.6% among progeny of wild Winnipeg River or Nelson River lake sturgeon and occurred at 2 geographically separate facilities. Namao virus was detected in almost 94% of the moribund or dead lake sturgeon according to a conventional polymerase chain reac- tion (cPCR) test that is based upon amplification of a 219 bp fragment of the virus major protein (MCP). The virus itself was large (242 to 282 nm) and icosahedral-shaped with 2 and a condensed bar-shaped core. It was found in virus factories within the host cell cytoplasm and displayed a tropism for the integument. Namao virus caused cellular changes characterized by enlarged eosinophilic epithelial cells in the gills and skin. Samples suspected of containing Namao virus did not have cytopathic effects on primary lake sturgeon or established white stur- geon cell lines. However, viral nucleic acid was detected in the former after prolonged incubation periods. Using primers designed from conserved regions of the MCP from NCLDVs, an estimated 95 to 96% of the Namao virus MCP open reading frame was captured. Phylogenetic analysis using the MCP of Namao virus and 27 other NCLDVs suggested that Namao virus and white sturgeon share a common evolutionary past and might be members of the family or a new, as yet unrecognized, virus family.

KEY WORDS: Namao virus · Mimiviridae · Endangered species

Resale or republication not permitted without written consent of the publisher

INTRODUCTION and , they form a putative monophyletic class of referred to as nucleo-cytoplasmic Viruses classified within the families Mimiviridae, large DNA viruses (NCLDVs; Table 1) (Iyer et al. and as well as the Mar- 2001, 2006, Koonin & Yutin 2010). A common ances- seillevirus and megaviruses are a ubiquitous compo- tral virus is suggested to have possessed a set of at nent of the aquatic environment (Chen et al. 1996, least 47 conserved genes that can be mapped onto Fuhr man 1999, Monier et al. 2008, Van Etten et al. the of extant NCLDVs (Koonin & Yutin 2010). Along with members of the families 2010). As a group, they share 5 common protein-

*Email: [email protected] © Inter-Research and Fisheries and Oceans Canada 2013 · www.int-res.com 196 Dis Aquat Org 102: 195–209, 2013

Table 1. Members of the nucleo-cytoplasmic large DNA virus (NCLDV) super-family

Virus taxon Host range Capsid size (kb) diameter (nm)

Iridoviridae Insects, cold blooded vertebrates 100−220 120−350 Ascoviridae Insects, mainly Noctuids 150−190 130 Asfarviridae Mammals 170 170−190 Poxviridae Insects, reptiles, birds, mammals 130−380 200 Phycodnaviridae Green algae; algal symbionts of paramecia & hydras 150−400 130−200 Amoeba 370 250 Mimiviridae CroV Marine microzooplankton 730 300 Amoeba 1181 390−400 Mamavirus Amoeba 1191 Not available Megaviruses Unknown 1259 440 Sturgeon viruses Namao virus (NV) Lake sturgeon Unknown 225−263 White sturgeon iridovirus (WSIV) White sturgeon Unknown 262 Missouri River sturgeon iridovirus (MRSIV) Pallid and shovelnose sturgeon Unknown 254 Russian sturgeon iridovirus (RSIV) Russian sturgeon Unknown 283

coding sequences (CDSs), including the major capsid geon Scaphirhynchus albus and shovelnose sturgeon protein (MCP), with an additional 177 shared by 2 or S. platorynchus (Kurobe et al. 2010, 2011). more families within the NCDLV super-family (Yutin Sturgeon NCLDVs can assume a complex lifecycle. et al. 2009). In some instances they cause upwards of 95% mor- NCLDVs replicate in the cytoplasm of the host cell tality within captive populations whereas in others or start their life cycle in the host nucleus and com- they are associated with a chronic debilitating wast- plete it in the cytoplasm (Iyer et al. 2001, 2006, Koonin ing syndrome, resulting in severely impaired growth & Yutin 2010). They share common biophysical char- and reduced survival of fry and fingerlings (Hedrick acteristics such as complex, isometric virion structures et al. 1990, LaPatra et al. 1994, Kurobe et al. 2011). (120 to 440 nm) and very large double-stranded DNA Outbreaks of in white sturgeon as well genomes (0.1 to 1.3 Mb) (Table 1). The number of as pallid or shovelnose sturgeon are typically ob - CDSs of many NCLDVs exceeds that of the small - served during the first year of growth in hatcheries est free-living Mycoplasma genitalium (Hedrick et al. 1990, 1992, Kurobe et al. 2011). Adults (~470 CDS, Claverie et al. 2006). The large repertoire can be refractory to clinical disease, but are likely of genes encoding proteins involved in DNA replica- subclinical carriers that may intermittently shed virus tion, processing, maturation and packaging, tran- (Hedrick et al. 1990, LaPatra et al. 1994, Kurobe et al. scription initiation and elongation as well as capsid 2011). Horizontal transmission of WSIV and MRSIV proteins and chaperones for assembly provide from convalescent to naïve juvenile sturgeon has NCLDVs with relative autonomy from the host cell. It been demonstrated (Hedrick et al. 1990, Drennan et is perhaps not surprising that they can have diverse, al. 2006, Kurobe et al. 2011) and there are sugges- complex life-cycles and a broad host range that in- tions that egg-associated transmission from adult to cludes amoeba, algae, insects, reptiles, birds, mam- offspring occurs during spawning (Hedrick et al. mals, and fish (Van Etten et al. 2010, Van Etten 2011). 1992, LaPatra et al. 1994, Georgiadis et al. 2001). Several NCLDVs of sturgeon (Acipenseridae) hith- Handling, stocking density and fluctuations in water erto recognized as unclassified members of the family levels, flow rates, and/or temperature are among the Iridoviridae, pose a potential disease risk for strategies other risk factors known to contribute to the onset designed to aid the recovery of threatened sturgeon and severity of these disease outbreaks (LaPatra et populations in North America. These include the al. 1994, 1996, Watson et al. 1998a, Georgiadis et al. white sturgeon iridovirus (WSIV), found in wild and 2000, 2001, Drennan et al. 2005, 2006). cultured white sturgeon Acipenser transmontanus WSIV and MRSIV have historically been diag- (Hedrick et al. 1990, LaPatra et al. 1994, Raverty et al. nosed using a combination of histology and electron 2003), as well as the Missouri River sturgeon iridovirus microscopy. Both viruses localize to the integument (MRSIV), which has been isolated from pallid stur- of the oropharynx and olfactory organs as well as the Clouthier et al.: Lake sturgeon Namao virus 197

fins and in some cases the epithelium of the gills tional (cPCR) and/or quantitative PCR (qPCR) assays (Hedrick et al. 1990, Watson et al. 1998b, Drennan et have been developed to detect WSIV or MRSIV. The al. 2007, Kurobe et al. 2011). Microscopic signs of current white sturgeon end-point or qPCR assays tar- infection include hypertrophied, basophilic or ampho- get viral DNA encoding the MCP (Kwak 2006, Kwak philic- to eosinophilic-staining cells and eccentric et al. 2006) which is one of the 5 orthologous DNA nuclei, as well as inclusion bodies containing isomet- sequences common to NCLDVs (Yutin et al. 2009), ric, doubly encapsidated virus particles displaying a while the MRSIV qPCR assay is based on amplifica- condensed bar-shaped nucleoid (Hedrick et al. 1990, tion of DNA encoding the serpin protein (Kurobe et 1992, Kurobe et al. 2011). A number of white stur- al. 2010). Neither of these assays is capable of detect- geon cell lines have been developed that are permis- ing viral DNA from the aforementioned NCLDV sive to WSIV amplification but in an isolate-specific found in Manitoba lake sturgeon. manner and sometimes without evidence of cyto- In this paper we provide the first description of a pathic effect (CPE) (Hedrick et al. 1991, Watson et al. new NCLDV found in populations of Manitoba (MB) 1998b). These as well as pallid and shovelnose- lake sturgeon Acipenser fulvescens, a species classi- derived cell lines do not support the growth of fied as endangered by the Committee on the Status MRSIV (Kurobe et al. 2011). More recently, conven- of Endangered Wildlife in Canada (COSEWIC 2011). Further, we provide evidence of a new NCLDV lineage comprised of these sturgeon viruses and discuss their phy- logenetic relationship to other viruses in the NCLDV clade.

MATERIALS AND METHODS

Case history

Unusual mortalities occurred in groups of juvenile lake sturgeon reared at the Grand Rapids Hatchery (GRH) (Grand Rapids, MB) and the University of Manitoba Aquatic Animal Holding Facility (UMAAHF) (Winnipeg, MB) (Fig. 1) in 2009 and 2010. The lake sturgeon reared at UMAAHF were derived from multiple crosses of eggs and sperm collected from sexually mature, wild Winnipeg River (MB) lake sturgeon (16 males, 8 females) captured at Slave Falls in May 2008. The fertilized eggs were hatched at the temporary Pinawa Hatchery (Pinawa, MB) and the fry (15 to 20 mm in length) were subse- quently transported on June 12, 2008 to UMAAHF. Tissue samples were col- lected for diagnostic evaluation on De- cember 7, 9, 11 and 17, 2008 from 7 ap- parently healthy lake sturgeon housed in 6 different tanks. On December 22, 2008, after a series of transfers to succes- Fig. 1. Location of rivers in Manitoba (dark grey shading), Canada where wild sively larger tanks, approximately 1758 lake sturgeon Acipenser fulvescens broodstock were collected and hatcheries where progeny were held. UMAAHF: University of Manitoba Aquatic Animal lake sturgeon (mean body weight 5.7 g, Holding Facility length not recorded) were placed in four 198 Dis Aquat Org 102: 195–209, 2013

600 gallon tanks receiving de-chlorinated 16°C mu- males) on July 9, 2009. The 2 populations of fish were nicipal water at 1 to 2 l min−1. In February 2009, mor- held separately in long, shallow troughs (L × W × D ≈ talities occurred amongst 606 lake sturgeon (mean 3.7 × 5.2 × 3.1 m) receiving untreated, ambient tem- body weight 3.0 g, cumulative percent mortality perature surface water from the Sas kat chewan River [CPM] 64%) held in Tank 600-1 (Fig. 2A). Then in (Cross Bay, Cedar Lake) (average temperature 10.1°C May 2009, mortalities were observed in Tanks 600-3, June through September, 4.9°C October through 600-4 and 600-5 stocked with 569, 195 and 388 lake May). Mortalities began to increase among approxi- sturgeon (mean body weight 19.3 g, mean fork length mately 1974 Nelson River lake sturgeon (Nov 4, 2009: 174 mm), respectively. CPM in these 3 tanks ranged mean body weight 17.3 g, mean fork length 15.5 cm) from 62 to 99%. Tissue samples from dead or mori- in late September 2009 and reached CPM of 99.6% bund fish were submitted for diagnostic evaluation. by February 19, 2010 (Fig. 2B). Similarly, mortalities The GRH received fertilized eggs derived from began to in crease among approximately 14 608 Win- wild Nelson River (MB) lake sturgeon (6 males, 4 fe- nipeg River lake sturgeon (mean body weight 11 g, males) on June 15, 2009 and fry from wild Winnipeg mean fork length 13.9 cm) in early November 2009 River (Pointe du Bois) lake sturgeon (17 males, 5 fe- and reached a CPM of 74% on May 28, 2010 at which time the remaining fish were euthanized. Tissue 100 samples were submitted from each group of lake 90 A sturgeon for diagnostic evaluation. 80 The UMAAHF also received fry derived from the 70 * wild Winnipeg River (Pointe du Bois) lake sturgeon 60 broodstock described in the previous paragraph. The 50 fish were received on October 8, 2009 and held in 40 600 gallon tanks receiving dechlorinated 16°C muni- 30 cipal water at 1 to 2 l min−1. Sporadic mortality oc- 20 curred in these tanks throughout 2010 (body weight 10 range 11 to 32 g, lengths not recorded) and tissue 0 samples from dead or moribund fish were submitted 1 3 5 7 91113151719212325272931333537394143454749 for diagnostic evaluation. Time (d) 100 90 B 80 Bacteriology, , histology 70 and electron microscopy Cumulative mortality (%) 60 50 Gill, spleen and kidney tissues from a small num- 40 ber of dead or moribund juvenile lake sturgeon col- 30 lected during the mortality events at UMAAHF (n = 20 10, Winnipeg River stock) or GRH (n = 15; 10 Win- 10 nipeg River stock, 5 Nelson River stock) were tested 0 for the presence of bacteria by inoculation of tryptic soy agar (TSA) supplemented with 5% sheep blood

6/19/09 7/31/098/21/09 2/10/09 4/12/09 1/15/105/02/102/26/103/19/109/04/104/30/105/21/10 10/07/09 11/09/09 10/23/0911/13/09 12/25/09 and MacConkey agar followed by incubation at 22°C Date and 35°C under aerobic as well as anaerobic condi- Fig. 2. Acipenser fulvescens. Cumulative mortality among ju- tions. Gill tissue was also used to inoculate chocolate venile lake sturgeon. (A) Sturgeon held at University of Man- agar as well as TSA containing 5% sheep blood fol- itoba Aquatic Animal Holding Facility: progeny (2008 year lowed by incubation in a CO2 candle jar at 22°C and class) from wild Winnipeg River broodstock collected at 35°C. Further, skin, gill and intestinal wet mounts as Slave Falls. Time zero was normalized to 16 d prior to onset of significant mortality within each of 4 tanks: (R) Tank 600-1, well as Gram stains of kidney and gill smears were Day 16 = Feb 11, 2009; (J) Tank 600-3, Day 16 = May 9, 2009; performed on samples from 8 fish from UMAAHF (M) Tank 600-4, Day 16 = May 27, 2009; (×) Tank 600-5, Day (Winnipeg River stock) and 15 fish from GRH (10 16 = May 12, 2009. (*) Date when fish remaining in tanks Winnipeg River stock, 5 Nelson River stock). 600-1 and 600-3 were removed. (B) Sturgeon held at Grand Barbells, gill arches, operculum, head skin, anal Rapids Hatchery: progeny (2009 year class) from (R) feral Nelson River broodstock collected near Landing River and and/or pectoral fins as well as kidney, liver, spleen (J) Winnipeg River broodstock collected at Pointe du Bois and/or pyloric caeca tissues were aseptically collected Clouthier et al.: Lake sturgeon Namao virus 199

from 33 Winnipeg River and 15 Nelson River lake stur- washed 5 times with sterile phosphate buffered geon and held at 4°C prior to analyses by virology on saline (PBS), minced into smaller pieces and then re- epithelioma papulosum cyprini (EPC), Chinook suspended in 0.25% porcine trypsin/EDTA (HyClone). salmon embryo 214 (CHSE-214), white sturgeon skin Cells were dispersed from the tissue fragments by (WSSK-1), white sturgeon spleen (WSS-2), lake stur- mechanical agitation for 20 min at 22°C. The result- geon gill (LSGI) and/or lake sturgeon gonad (LSGO) ing cell suspension was diluted 1:1 (v/v) with Dul- cell lines (Fijan et al. 1983, Lannan et al. 1984, Hedrick becco’s Modified Eagles Medium (DMEM)/Ham’s F- et al. 1991). Some of these samples were also submit- 12 supplemented with 10% FBS, 200 IU penicillin ted to the British Columbia Freshwater Fisheries Soci- and 200 µg streptomycin ml−1 (Life Technologies), ety (BCFFS) (Duncan, British Columbia). Each cell line centrifuged at 1228 × g for 15 min at 4°C and then was propagated at 16°C in minimum essential medium resuspended in fresh media prior to being trans- with Earle’s salts (MEM-E) or Hanks salts (MEM-H) ferred to 25 cm2 flasks. The lake sturgeon cell mono- supplemented with 10% fetal bovine serum (FBS) and layers were propagated at 16°C in MEM-H supple- 2 mM L-glutamine (Life Technologies). Replicate mented with 10% FBS, 2 mM L-glutamine and 2× wells of a 24-well tissue culture plate containing cell antibiotic/antimycotic (Life Technologies). monolayers were inoculated with 0.1 ml of a 1:50 dilu- tion of clarified homo genate generated from pools of tissue from a single fish. After an absorption period of DNA synthesis and plasmid purification 1 h, 1 ml of MEM-E or MEM-H supplemented with 2% FBS (MEM-E2, MEM-H2), 2 mM L-glutamine and DNA representing the gene encoding the WSIV 1× antibiotic/antimycotic (Life Technologies) was MCP (Genbank accession number DQ897645) was added to each well of the plate, the plate was incu- synthesized and ligated into the cloning vector bated at 16°C and the monolayers were observed pJ204 (DNA2.0). The construct was then trans- daily for evidence of CPE. At the time of harvest, each formed into Escherichia coli DH5α competent cells inoculated monolayer was scraped from the bottom of and plasmid DNA was purified with a QIAprep Spin the well into the culture fluid and the cell suspension Miniprep Kit (Qiagen) according to the manufac- was transferred to a storage vial and placed at –80°C. turer’s instructions. In preparation for histological examination, an inci- sion was made in the abdomen of 9 moribund lake sturgeon from the Winnipeg River stock (5 from DNA extraction GRH, 4 from UMAAHF) and 5 from the Nelson River stock and whole fish were placed in 10% buffered Tissue samples including ventral head skin, ab- formalin. After 24 to 48 h, portions of the fixed tissues dominal skin, gill arches, barbells, operculum, kidney, were processed for light microscopy into paraffin liver as well as pectoral and anal fins were collected embedded blocks, sectioned at 5 µm, deparaffinized from dead, moribund or frozen lake sturgeon from and then stained with hematoxylin and eosin (H&E). mortality events occurring at GRH and UMAAHF. For transmission electron microscopy (TEM), for- Homo gen ates of 50 mg portions of these tissues were malin-fixed gill tissue from a single juvenile Win- generated in lysis buffer (Qiagen buffer ATL) using a nipeg River lake sturgeon (MAAHF) was rinsed in 5 mm stainless steel bead (Qiagen) and TissueLyser buffer and post-fixed in 1% aqueous osmium tetrox- (Qiagen) operated twice for 2 min at 30 Hz. DNA was ide, dehydrated through a graded ethanol series, extracted from the tissue homogenates using the infiltrated and then embedded in Araldite 502/ DNeasy Blood and Tissue Kit (Qiagen) as outlined by Embed 812 resin. Thin sections (about 100 nm) were the manufacturer and then quantified using the Nano - stained with uranyl acetate and lead citrate prior to drop 8000. being examined using a Philips CM 120 transmission electron microscope at 80 kV. Digital imaging was performed with an AMT XR-611 camera system. PCR amplification, cloning and DNA sequence analysis

Establishment of lake sturgeon cell lines DNA samples were tested for the presence of WSIV and MRSIV using qPCR protocols described by Kwak Gill and gonad tissues were aseptically removed (2006) and Kurobe et al. (2010), respectively. Sam- from 2 juvenile lake sturgeon (52.5 and 62.7 g), ples were also submitted to the Animal Health Cen- 200 Dis Aquat Org 102: 195–209, 2013

ter (AHC) in Abbotsford, BC for additional molecular Table 3. Oligonucleotides used in the present study for testing. amplification of DNA encoding lake sturgeon Namao virus major capsid protein (partial). Nucleotide positions are Primers targeting the Namao virus MCP were shown relative to white sturgeon iridovirus major capsid designed based on conserved regions identified from protein coding region (GenBank accession no. DQ897645) sequence alignments of the MCP from representa- tive members of the NCLDV family of viruses Primer Sequence Nucleotide (Table 2). Portions of the Namao virus MCP gene (5’ to 3’) position (bp) were amplified by cPCR using pairwise combinations of the primers listed in Table 3 and DNA was speF CCA GAA ATG ACT TAC TTC AAG 64 extracted from tissue samples collected from popula- ginF GGT ATC AAC GTA TAT TCG TTT GC 1369 ginR GCA AAC GAA TAT ACG TTG ATA CC 1391 tions of lake sturgeon experiencing mortality at pekR CCA GAA GGT TGG TGC TTT TCA GG 1421 either GRH or UMAAHF. The origin of the amplicon glaR GTA TGC CAG ACC CGC TAG ACC 1587 was tested using DNA from Namao virus-infected

Table 2. Major capsid protein of representative members of the nucleo-cytoplasmic large DNA virus (NCLDV) superfamily. Capsid NCLDV domains were identified by the Conserved Domain Database (see ‘Materials and methods’)

Virus Abbre- Gene Protein NCLDV Accession number Reference viation (bp) (aa) domains DNA Protein

Unclassified mimivirus-like Namao virus NV 1524 508 2 JX155659 Present study partial partial White sturgeon iridovirus WSIV 1596 531 2 DQ897645 ABK34555.1 Direct submission Mimiviridae polyphaga APMV-1 1782 593 2 NC 014649.1 YP003986929 Legendre et al. (2011) mimivirus (Bradford isolate) Acanthamoeba polyphaga APMV-2 1776 591 2 JN036606.1 AEJ34665.1 Boyer et al. (2011) mimivirus (M4 isolate) Cafeteria roenbergensis virus CroV 1521 506 2 NC 014637.1 YP003969975.1 Fischer et al. (2010) Megavirus chiliensis MVChile 1770 589 2 NC 016072.1 YP004894515.1 Arsian et al. (2011) Megavirus courdo MVCour 1698 565 2 JN885991.1 AEX61606 Direct submission Acanthamoeba castellanii mamavirus ACMV 1776 591 1 JF801956.1 AEQ60611.1 Colson et al. (2011) Phycodnaviridae Organic lake phycodnavirus 2 OLPV-2 1611 536 1 HQ704803.1 ADX06358 Yau et al. (2011) Phaeocystis pouchetii virus PPV 1563 520 1 EU006631.1 ABU23715.1 Larsen et al. (2008) Chrysochromulina ercina virus CEV 1755 584 1 EU006628.1 ABU23712.1 Larsen et al. (2008) Pyramimonas orientalis virus POV 1191 396 1 EU006630.1 ABU23714 Larsen et al. (2008) Heterosigma akashiwo virus HAV 1323 440 1 AB198422.1 BAE06835 Nagasaki et al. (2005) Acanthocystis turfacea chlorella virus ATCV 1317 438 1 NC 008724.1 YP001426761.1 Direct submission Paramecium bursaria chlorella virus PBCV 1314 437 1 NC 009898.1 YP001497813.1 Fitzgerald et al. (2007) Micromonas pusilla virus MPV 1257 418 1 HQ633072.1 AET43572 Direct submission Ostreococcus tauri virus OTV 1269 422 1 NC013288.1 YP003495004.1 Weynberg et al. (2009) Feldmannia spp. virus FSV 1308 435 1 NC011183.1 YP002154681.1 Schroeder et al. (2009) Ectocarpus siliculosus virus ESV 1428 475 1 FN648730.1 CBN80416.1 Cock et al. (2010) Iridoviridae Chilo iridescent virus CIV 1404 467 1 NC003038.1 NP149737 Jakob et al. (2001) Invertebrate iridescent virus 3 IIV3 1401 466 1 NC008187.1 YP654586 Delhon et al. (2006) Frog virus 3 FV3 1392 463 1 FJ459783.1 ACP19256.1 Holopainen et al. (2009) Infectious spleen and kidney virus ISKNV 1362 453 1 AF371960.1 AAL98730.1 He et al. (2001) Lymphocystis disease virus LDV 1380 459 1 EF103188.1 ABL07488.1 Direct submission Unclassified Marseillevirus MV 1434 477 1 NC013756.1 YP003407071.1 Boyer et al. (2009) Ascoviridae Diadromus pulchellus ascovirus 4a DPAV 1305 434 1 AJ312705.1 CAC84483.1 Stasiak et al. (2003) Asfarviridae African swine fever virus ASFV 1941 646 1 XM369245.2 XP369245.2 Direct submission Clouthier et al.: Lake sturgeon Namao virus 201

lake sturgeon tissue, naïve lake sturgeon tissue as (Guindon et al. 2010). The Baye sian trees were esti- well as white and lake sturgeon cell lines. Each 25 µl mated using multiple independent runs of aamodelpr reaction contained 1× PCR buffer (Applied Biosys- = mixed and default settings for 1 500 000 genera- tems), 1.5 mM MgCl2, 200 µM deoxynucleoside tri- tions when the average deviation of the split fre- phos phates, 1.25 U AmpliTaq Gold DNA polymerase quencies was <0.002. For maximum-likelihood trees, (Applied Biosystems), 0.8 µM of each primer and clade confidences were estimated from 1000 boot- between 100 to 500 ng of DNA. Thermocycling strap replicates. The output tree produced from each parameters were as follows: 95°C for 5 min, 40 cycles method was visualized using FigTree v1.3.1 software of (95°C for 30 s, 52 or 53°C for 30 s, 72°C for 1 or (Rambaut 2008). 1.5 min) followed by a post-cycle extension at 72°C for 10 min. Amplicons generated from 25 to 50 µl of the cPCR RESULTS product were analyzed for purity and size by electro- phoresis in 1% agarose gels, purified with the Disease signs and bacteriology QIAquick Gel Extraction Kit (Qiagen) as outlined by the manufacturer and then TA cloned into vector Lake sturgeon mortality events at GRH or pGEM-T Easy (Promega). Plasmid DNA was isolated UMAAHF during 2009 to 2010 (Fig. 2) were charac- from cultures of transformed E. coli as outlined above terized by fish inappetence, anorexia and erratic and screened for inserts by cPCR using the relevant behavior such as swimming with the tail up and head primer pairs. Both strands of DNA from at least 3 pos- down in the water column, unstable equilibrium and itive clones per amplicon were sequenced by the gasping near the surface of the water. Petechial dideoxynucleotide chain termination method using haemorrhaging was present at the base of the pec- an automated sequencer (Sanger et al. 1977). Analy- toral and anal fins as well as around the mouth area ses of the DNA sequences were performed using in some of the fish. Dark red gills with pinpoint foci, BioEdit v7.0.9.0 software (Hall 1999). accumulations of gill mucus and exaggerated gill movement were also noted. However, bacterial growth was not observed on media inoculated with BLASTP, sequence alignment and samples derived from moribund fish. phylogenetic analysis

Sequences displaying similarity to the deduced Virology and PCR amino acid sequence of the partial Namao virus MCP were identified from protein databases searched Namao virus was detected by cPCR amplification using the BLASTP program (Altschul et al. 1990, of a 219 bp MCP DNA fragment in almost 94% (59 of 1997). Protein sequence alignments were performed 63) of those samples in this study derived from dead using T-Coffee (Notredame et al. 2000, Di Tommaso or moribund lake sturgeon originating from the et al. 2011) with sequences that were trimmed to the Nelson or Winnipeg Rivers (Table 4). The ginF and first amino acid aligning with the N terminal end of the Namao virus MCP fragment and the last amino acid at the C terminal end. A percent identity and Table 4. Diagnostic test results (no. positive/no. tested) from similarity matrix for the trimmed MCP sequences samples taken during disease outbreaks in the 2008 and 2009 year classes of juvenile lake sturgeon, sourced from the was created using the Matrix Global Alignment Tool Winnipeg (WR) and Nelson River (NR), and raised at Grand (MatGAT 2.02) software (Campanella et al. 2003). Rapids Hatchery (GRH) and the University of Manitoba Conserved domains in the truncated protein se quen - Aquatic Animal Holding Facility (UMAAHF). cPCR: con- ces were annotated through the Conserved Domain ventional polymerase chain reaction; qPCR, quantitative PCR. See Tables 1 & 2 for virus abbreviations Database using the National Center for Biotechnol- ogy Information’s (NCBI) Conserved Domain Search Molecular 2008 2009 service (Marchler-Bauer et al. 2011). test method UMAAHF GRH UMAAHF Phylogenetic tree reconstructions were estimated WR NR WR WR by 2 methods: Bay e sian inference analysis as imple- mented by MRBAYES v3.2.1 (Huelsen beck & Ron- NV cPCR 7/8 3/3 35/37 14/15 WSIV qPCR 0/8 0/3 0/35 0/12 quist 2001, Ronquist & Huelsenbeck 2003) and maxi- MRSIV qPCR 0/8 0/3 0/35 0/12 mum-likelihood as implemented by PHYML 3.69 202 Dis Aquat Org 102: 195–209, 2013

glaR primer pair yielded a cPCR product of both the the MRSIV serpin-based qPCR tests were able to expected size (data not shown) and sequence detect Namao virus in the same samples. Our Namao (described below). Neither the WSIV MCP-based nor virus MCP-based cPCR test, which also detects WSIV (including artificially synthesized DNA encoding the WSIV MCP), MRSIV and a recently diagnosed virus A from white sturgeon cultured in British Columbia, will be described in detail elsewhere. The 7 samples collected in December 2008 prior to the 2009 out- break at UMAAHF tested negative for the presence of Namao virus nucleic acid using the ginF/glaR cPCR assay. Inoculation of primary lake sturgeon cell lines (LSGI or LSGO) with tissue homogenates shown to be positive for Namao virus by cPCR did not result in noticeable CPE after 21 to 60 d of observation or blind passage on the same cell lines. However, some of the culture fluid containing suspended lake stur- geon cells from CPE-negative monolayers tested pos- 10 µm itive for Namao virus after the first passage (the sec- ond passage was not tested by cPCR). The cells from B white sturgeon (WSSK-1, WSS-2), Chinook salmon Oncorhynchus tshawytscha (CHSE-214) and com- mon carp Cyprinus carpio (EPC) did not display CPE within 21 d of inoculation with tissue samples derived from the same populations of lake sturgeon. The WSS-2 cells, inoculated with 4 samples, 2 of which tested positive for Namao virus by cPCR, tested negative by the same assay for the presence of Namao virus after a single passage. The cPCR did not amplify the 219 bp DNA product from any of the 10 µm naïve cell lines used in this study.

C Histology and TEM

Low numbers of hypertrophic cells were found in H&E stained histological samples of gill and skin epi- thelia tissues from moribund lake sturgeon (Fig. 3). The cytomegalic cells were scattered throughout the epithelial layer of the gill lamellae and appeared to contain granular, cytoplasmic inclusion bodies often associated with iridovirus-like infections of white-, pallid- and shovelnose sturgeon. Histological sec- tions from the same fish did not show evidence of sig- nificant changes in kidney, heart, brain lower intes- 10 µm tine, pancreas or nervous tissues. Enumeration of Fig. 3. Acipenser fulvescens. Light photomicrographs of inclusion bodies in longitudinal sections of 14 juve- H&E stained epithelial tissue sections from moribund juve- nile lake sturgeon collected from GRH in May 2009 nile lake sturgeon. (A) Skin tissue from Winnipeg River lake indicated a tropism of the virus for epithelial cells of sturgeon (2008 year class) housed at the University of Mani- the integument (Table 5). toba Aquatic Animal Holding Facility. (B) Skin and (C) gill Examination of cytomegalic cells within lake stur- tissue from Winnipeg River lake sturgeon (2009 year class) housed at Grand Rapids Hatchery. Large amphophilic intra- geon gill lamellae epithelium by TEM revealed the cytoplasmic inclusions are evident (arrows) presence of icosahedral-shaped virus particles in the Clouthier et al.: Lake sturgeon Namao virus 203

Table 5. Acipenser fulvescens. Enumeration of inclusion cell cytoplasm (Fig. 4). Some of the virions contained bodies (average no.) in longitudinal sections of moribund or a condensed bar-shaped core. Amorphous virus-like mildly infected juvenile lake sturgeon from the Winnipeg River (WR) and Nelson River (NR). ND: not done particles as well as empty or incomplete capsids were also evident. Intact virions had 2 capsids and were Site Average number of inclusion bodies approximately 242 nm in diameter as measured from Moribund sturgeon Juvenile sturgeon side to side and 282 nm vertex to vertex (Fig. 4, WR NR WR Table 1). (n = 5) (n = 4) (n = 5)

Nasal pits 8.5 4 1 Snout 3.5 2.25 0 MCP alignment and analysis Mouth 4.3 1.75 0 Gill 3.6 3.5 0.4 We captured an estimated 95 to 96% of the Namao Epidermis virus MCP open reading frame or its inferred amino Cranial 14.1 8.45 2.0 Abdominal - mid 9.4 3.5 0.8 acids based upon comparison with the WSIV MCP Abdominal - distal 4.5 ND 0.5 sequences (Fig. 5, Table 2; GenBank accession num- Fins ber JX155659). The identity of the amino acids Pectoral 12.3 2.75 0 shared between Namao virus and WSIV was 83.5% Dorsal 17 ND ND Pelvic 4.5 3.5 0 (Table 6). Namao virus and WSIV MCP sequences shared the highest E-value (7e-99) with viruses grouped in the Mimiviridae family and to a lesser extent Phycodna viridae (3E-46) and Iridoviridae (1e- 15) families, according to BLASTP analysis (Altschul et al. 1990, 1997). Conserved domains identified within the Namao virus MCP are presented in Fig. 5.

Phylogenetic analyses

Namao virus and WSIV grouped more closely to each other than to any of the other viruses based upon phylogenetic analysis using the MCP amino acid sequence (Fig. 6). This was the case for tree reconstructions employing Bayesian inference (Huel - senbeck & Ronquist 2001, Ronquist & Huelsenbeck 2003) or maximum-likelihood methods (Guindon et al. 2010). The sturgeon viruses appeared in the Mimi - viridae and Phycodnaviridae lineage but partitioned into the Mimiviridae clade. Both sturgeon viruses were clearly separate from and were not members of the Irido-Asco-MV clade. Within the Mimiviridae family, the 2 megaviruses (proposed genera) and clustered together and shared a common ancestor with the mamavirus, CroV and sturgeon viruses. While this relationship was similar using either method of tree reconstruc- tion, the relative branching topography for the latter Fig. 4. Acipenser fulvescens. Transmission electron micro- graphs of cytoplasmic hexagonal virus particles in epithelial 3 viruses was different. In the maximum-likelihood cells of gill lamellae from Winnipeg River, juvenile lake stur- tree, CroV and mamavirus formed a lineage together geon (2008 year class) held at the University of Manitoba that split from the sturgeon viruses after the node Aquatic Animal Holding Facility. Intact virions were 225 to representing the ancestral Mimiviridae progenitor. 263 nm (n = 18, mean ± SD = 242 ± 11 nm) in diameter, as measured from side to side, and 263 to 300 nm from vertex to However, the bootstrap values were too low (221/ vertex (n = 18, mean ± SD = 282 ± 13 nm). Scale bars: (A) 1000) to resolve the phylogenetic position of the stur- 500 nm; (B) 100 nm geon viruses relative to CroV and mamavirus. 204 Dis Aquat Org 102: 195–209, 2013 Clouthier et al.: Lake sturgeon Namao virus 205

Fig. 5. Alignment of major capsid protein (MCP) sequences from 8 viruses found to cluster together in the phylogenetic tree presented in Fig. 6. The MCP sequences were trimmed to the first and last amino acids aligning with the N and C terminal ends, respectively, of the Namao virus (NV) MCP fragment targeted by primers from the present study. Identical amino acid residues shared by 2 or more sequences are shaded black whereas similar residues are shaded grey. The numbers preceding and following the alignments indicate the positions of the first and last residues of the aligned regions in the corresponding protein sequences. Bars above the sequence delineate the conserved nucleo-cytoplasmic large DNA virus domains identified in the NV MCP through the Conserved Domain Database and National Center for Biotechnology Information’s Conserved Domain Search service. The beta strands predicted in the first and second jelly-roll motifs are designated B1 to I1 and B2 to I2, respectively. The 8 conserved capsid domains reported by Larsen et al. (2008) for viruses classified as members of family Phycodnaviridae are boxed and labelled I to VIII. Virus abbreviations are defined in Table 2

Table 6. Analysis of the major capsid protein from Namao virus and other nucleo-cytoplasmic large DNA viruses. Data are values or ranges of % identity/% similarity of amino acid sequences. WSIV: white sturgeon iridovirus; IAMV: Irido-asco- Marseilleviruses

Virus taxon/clade Sturgeon viruses Mimi-mama- Phycodnaviruses IAMV Namao virus WSIV megaviruses

WSIV 83.5 94.1 Mimi-mama-megaviruses 20.6−34 20.8−32 22.1−99.1 41.6−54.7 40−54.1 43.5−99.1 Phycodnaviruses 20.5−29.8 19.1−30.2 20.3−42.3 25−77.4 40.2−53.2 38.4−51.9 37.4−60.0 39.1−86.7 IAMV 17.8−21.6 17.7−20.9 15.4−22.2 16.8−26 27.7−60.1 35.8−37.4 33.9−38.0 29.8−40.0 32.3−48.2 47.2−73.4 Asfarviridae 17.8 16.9 15.8−17.7 14.4−18 14.5−17.7 33.9 32.9 30.7−38.3 25.6−34.6 29−33.2

FV3 ISKNV DISCUSSION

IIV3 LDV We have discovered a new virus, Namao virus, as- CIV MV DPAV sociated with die-offs among juvenile lake sturgeon being reared as part of a conservation stocking pro- ASFV gram for endangered populations of sturgeon from the Nelson and Winnipeg rivers, MB. The ultrastruc- ture of Namao virus, including its relatively large size ESV FSV (242 nm), was consistent with those reported for WSIV, MRSIV and a virus from Russian sturgeon Acipenser guldenstadi (Hedrick et al. 1990, 1992, Ad- kison et al. 1998, Kurobe et al. 2011). The virus also OTV displayed a tropism for the integument of the body MPV POV and gills and caused cellular abnormalities consistent PBCV ATCV ACMV with pathognomonic changes used to presumptively HAV diagnose sturgeon NCLDVs. This information, in NV OLPV -2 conjunction with the phylogenetic relationship and CEV CroV WSIV PPV MVChile tree topology we observed, suggests that Namao MVCour virus and WSIV as well as other sturgeon NCLDVs APMV-1 APMV-2 (authors’ unpubl. results) share a common evolution- 0.4 ary past and might be members of the Mimiviridae or Fig. 6. Phylogenetic tree reconstruction of the major capsid a new, as yet, unrecognized virus family. protein (MCP) of 27 nucleo-cytoplasmic large DNA viruses, Further tests will be required to establish causality including Namao virus (NV) and white sturgeon iridovirus between infection with Namao virus and lake stur- (WSIV). The tree was generated using Bayesian inference geon morbidity and mortality. However, observations (BI) from MCP sequences with nodes labeled with BI poste- rior probabilities (Materials and methods). Abbreviations of in this study point to this effect and the link is coher- other viruses and accession numbers are shown in Table 2 ent with our general understanding of sturgeon 206 Dis Aquat Org 102: 195–209, 2013

NCLDVs. Apart from the pathognomonic changes (Slave Falls) or subsequently at Pinawa Hatchery we observed, the virus was present in almost all the (site of larval rearing) which used untreated water fish samples (93.6%) that were linked to the die-offs from the Winnipeg River. The source of the virus at and in a suitable condition for analysis by cPCR. Ini- GRH in the 2009 year class of Nelson River and Win- tial attempts to isolate the virus in pure culture on nipeg River is less clear and will require completion white sturgeon cell lines were unsuccessful. This is of our ongoing epidemiological study. In addition to similar to MRSIV (Kurobe et al. 2011) and some iso- the sources already noted, the virus at GRH could lates of WSIV (Hedrick et al. 1990, Drennan et al. have been introduced from the Nelson River lake 2006). We were able to detect Namao virus, although sturgeon or the Saskatchewan River which supplies weakly, by cPCR when suspect tissues were inocu- water to the hatchery. lated and then incubated for prolonged periods on The cumulative mortality in lake sturgeon popula- primary lake sturgeon cell lines but without evidence tions harboring Namao virus reached a high level, of CPE. This could mean that the cells supported approaching 100% in the 2009 Nelson River year virus replication or that the virus present in the orig- class held at GRH. This is similar to the level of mor- inal inoculum was being detected. The difficulty we tality caused by WSIV in white sturgeon or MRSIV in encountered culturing the virus has prompted our pallid or shovelnose sturgeon naturally exposed to interest in ascertaining whether Namao virus and the virus (Hedrick et al. 1990, 1992, Kurobe et al. related sturgeon NCLDVs might be amenable to 2011). Qualitatively, the kinetics of the die-offs were propagation on cultured amoebae similar to mem- distinct when comparisons were made between bers of the family Mimiviridae (La Scola et al. 2003, UMAAHF and GRH but the outcome was similar. 2008) or the unclassified Marseillevirus (Boyer et al. The die-offs at UMAAHF occurred over the course of 2009). This follows from the proposal that amoeba 2 to 4 wk whereas those at GRH occurred over the function as melting pots for these and possibly other course of approximately 1 mo in fish derived from the NCLDVs (Boyer et al. 2009). Successful culturing of Nelson River and 6 mo in the Winnipeg River stock. Namao virus would open a convenient avenue for Such variation in mortality kinetics is not without examination of the disease dynamics and enable ful- precedent among sturgeon NCLDV infections, and fillment of the criteria required to establish causality has been attributed to risk factors including hand- of disease (Koch 1890, Hill 1965, Evans 1976). ling, stocking density, and water conditions (LaPatra WSIV and MRSIV display signs of a complex life- et al. 1994, 1996, Watson et al. 1998a, Drennan et al. cycle that can manifest as an acute or chronic infec- 2005, 2006). Each of these factors was different tion in juvenile sturgeon depending on rearing con- between the 2 facilities as were the stocks of fish held ditions during the first year of growth (Hedrick et al. at GRH. Specific studies designed to assess the role 1990, 1992, LaPatra et al. 1994, 1996, Watson et al. or influence these variables have on Namao virus 1998a,b, Drennan et al. 2005, 2006, Kurobe et al. disease dynamics could be used to avoid or minimize 2011). Our observations suggest that Namao virus is outbreaks caused by the virus in hatcheries. Further, similar in this respect. We suspect that the die-offs at it will be important to control viral trafficking of UMAAHF and GRH signaled the switch between the Namao virus in lake sturgeon through the manage- chronic and acute phases of the virus life-cycle. In ment of factors related to virus source, prevalence this scenario, the fish could have been infected either and transmission much like what has already been horizontally or vertically at the time of fertilization or accomplished for WSIV in white sturgeon through subsequently by horizontal transmission prior to the Kootenai River White Sturgeon Conservation transport to UMAAHF. However, we cannot rule out Aquaculture Program (LaPatra et al. 1999, KTOI the possibility that filtered and dechlorinated munic- 2007). ipal water at UMAAHF was the source of infection in In the present study, 7 fish collected 2 mo prior to lake sturgeon at the facility, especially since Mar- disease onset at UMAAHF tested negative for seillevirus, mamavirus and mimivirus were all iso- Namao virus by cPCR. The absence of detection does lated from samples of water collected from cooling not rule out the presence of Namao virus in these towers in Europe (La Scola et al. 2003, 2008, Boyer et fish. Especially given the limited sensitivity of the test al. 2009). From a parsimonious standpoint, however, compared to qPCR, absence of verification by a dif- it seems more likely that the fish were infected prior ferent test method such as cell culture, and the possi- to transport to UMAAHF. This would point to infec- bility that the virus might persist at extremely low tion occurring in the 2008 year class of Winnipeg levels as a localized chronic infection in asympto- River lake sturgeon at the broodstock collection site matic fish. In addition, differences in the ability to Clouthier et al.: Lake sturgeon Namao virus 207

detect sturgeon NCLDVs might reflect the choice of Our results indicate that Namao virus and WSIV tissues sampled or differences in disease susceptibil- shared a common lineage with CroV and mamavirus ity of the host (Kwak et al. 2006, Drennan et al. 2007, within the Mimiviridae family. The close phylogenetic Kurobe et al. 2010). The outbreaks at UMAAHF were relationship between the latter 2 viruses was reported temporally asynchronous but acute, indicating that a previously by Fischer et al. (2010). The genome of large percentage of the fish were either harboring mamavirus is approximately 1.6-fold larger than that the virus at the onset of mortality or that the fish were of CroV. In this context, the sturgeon NCLDVs’ exposed to a high dose of virus on 4 separate occa- capsid diameters were most similar to that of CroV in sions. Again, the latter would indicate that an occa- size and were approximately half the size of that for sional large amount of virus was present in the megaviruses, the largest known members of the fam- treated municipal water or that a highly contami- ily Mimiviridae. The Namao virus and WSIV MCPs nated fomite was present and led to infection of fish both contained 2 conserved NCLDV capsid domains in the different tanks on 4 different occasions. None (Marchler-Bauer et al. 2011) separated by an insertion of these possibilities can be ruled out from having of approximately 135 or 91 amino acids, respectively occurred in the present study but, as we mentioned (Fig. 5, Table 2). This insertion was similar to that de- earlier, the presence of the virus in treated, munici- scribed for the APMV-1 capsid protein 1 (Azza et al. pal water seems unlikely, though not impossible. In 2009) and like the mimivirus, this insertion occurred fact, Drennan et al. (2006) reported that the acciden- in the predicted DE loop of the second jelly roll along tal introduction of as little as 100 ml of Kootenai River the sturgeon virus capsid proteins. The Namao virus water resulted in fish testing positive for WSIV dur- and WSIV MCPs also contained the 8 conserved cap- ing the course of an experiment conducted in tanks sid domains reported by Larsen et al. (2008) for receiving municipal water. viruses classified as members of the family Phycod- The classification of sturgeon NCLDVs might be - naviridae. The mamavirus, like most of the viruses come clearer with our phylogenetic analysis coupled belonging to the Phycodnaviridae and all of the irido- with commonalities observed among their life-cycles. asco-MV-asfarviruses listed in Table 2, did not con- The general topology of the phylogenetic tree we tain this insertion and instead had a single, fused con- reconstructed was strongly supported by Baye sian served capsid domain. Further, the mamavirus MCP inference analysis and was consistent with virtually contained an N-terminal insertion of 162 amino acids all those trees reported in the literature pertaining to between MCP Domains 1 and 2 of the phycodna - NCLDVs regardless of the method used to recon- virus-like capsid proteins. struct the tree or whether the tree was generated If Namao virus and related sturgeon viruses are with the MCP or concatamers of conserved NCLDV indeed members of the Mimiviridae family, then the protein sequences (Tidona & Darai 2000, Raoult et al. host range of this group of viruses would be extended 2004, Iyer et al. 2006, Larsen et al. 2008, Monier et al. from unicellular eukaryotes to lower vertebrates. Like - 2008, Boyer et al. 2009, Yutin et al. 2009, Fischer et al. wise, the names as well as the taxonomic grouping of 2010, Koonin & Yutin 2010, Arslan et al. 2011). It must suspected ‘irido-like’ sturgeon NCLDVs will likely be kept in mind that our phylogenetic tree was require amendment based on the results of this study. reconstructed using only the MCP, one of the 5 We believe this might be the case regardless of the nucleo-cytoplasmic virus orthologous genes (Yutin et final taxonomic designation of the sturgeon NCLDVs al. 2009). The genomes of these giant viruses possess because comparison of iridovirus MCP sequences to a complex repertoire of genes from various origins that of Namao virus and WSIV reveal fundamental dif- including viruses, bacteria and eukaryotes, and ferences in their evolutionary relationship. reflect a rich evolutionary history (Filée 2009) and it is possible that the genome of Namao virus and related sturgeon NCLDVs, aside from their MCP Acknowledgements. This work was supported in part by genes, are more distantly related to the Mimiviridae Manitoba Hydro. L. Burton generated the electron micro - scopy images and J. Neufeld identified icosahedral-shaped and Phycodnaviridae families than indicated by our particles as iridoviruses at the Canadian Food Inspection results. For instance, there is some evidence of gene Agency, National Centre for Foreign Animal Disease. S. ex change between the distantly related Marseille- Mead supplied the white sturgeon skin and spleen cell lines virus and mimivirus (Boyer et al. 2009). Our ongoing to S.C.C. and conducted some of the virological assays for the Freshwater Fisheries Society of BC (Duncan, BC) and J. meta geno mic studies in conjunction with the work of Robinson performed some of the molecular assays for the others may provide further insight into the taxonomy Animal Health Centre, Ministry of Agriculture (Abbotsford, of these viruses. BC). A. Lewin created the maps. 208 Dis Aquat Org 102: 195–209, 2013

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Editorial responsibility: V. Gregory Chinchar, Submitted: July 16, 2012; Accepted: October 25, 2012 Jackson, Mississippi, USA Proofs received from author(s): January 23, 2013